Toxoplasma gondii is an obligate intracellular parasite that infects nearly all mammalian and avian nucleated cells. Infection of immunocompetent individuals with T. gondii is generally asymptomatic;however, toxoplasmosis can have severe implications in fetuses and immunocompromised individuals, such as AIDS patients and organ transplant recipients. Virulence mutants have been generated and identified in a signature-tagged mutagenesis library. The proposed research will characterize mutants from the library that are less virulent and unable to cause a typical acute infection. The mutants will be used to outline a protective immunity profile and provide insight into possible drug targets to limit infection of T. gondii in individuals most at risk for severe toxoplasmosis. T. gondii virulence mutants identified in an in vivo screen will be complemented to confirm the gene causing the mutant phenotype. The expression level and location of the protein product of the virulence gene will be determined. A knockout mutant of the virulence genes will be constructed and used to immunize mice before injection of a lethal dose of T. gondii. Efficiency of protective immunity will be quantified by change in LD50 and the number of cysts per brain during the chronic phase of infection. The protective profile will be examined through measurements of cytokines induced in mice and dissemination of the parasites during infection. Toxoplasma gondii is a parasitic pathogen that can cause severe and life-threatening illnesses in fetuses and immunocompromised individuals, including AIDS patients and transplant recipients. The proposed research will utilize acute virulence mutants to examine protective immunity and provide insight into possible drug targets to protect these individuals from serious infection with T. gondii.